DESCRIPTION (Adapted from abstract): This renewal application proposes to test several hypotheses concerning the interplay between the strength of receptor-mediated inter-membrane adhesion and i) the molecular structure, ii) the state of protein aggregation, iii) thermodynamic and kinetic properties of the bonds, and iv) the rate of detachment. The focus is on the structural, thermodynamic and kinetic properties that determine the adhesive strength of homotypic cadherin interactions and heterotypic interactions made between the T-cell receptors, CD2 and CD48. The combination of direct surface force measurements, fluorescence dynamics spectroscopy and simulations will identify biophysical mechanisms that control protein-mediated adhesion between model cell membranes. The measured forces between cadherin extracellular domains as a function of their intermolecular separation will elucidate the inter-protein configurations and domain interactions responsible for recognition. These experiments will test current models for the role of cadherin structure in adhesion. Adhesive domains within the extracellular region of cadherin will be identified through the use of full length and deletion mutants of this region. The strength of receptor-mediated adhesion is postulated to depend on receptor-ligand dynamics and on the bond affinities. Adhesive strengths dictate cell migration speeds and the extent of cell spreading on surfaces. The proposed relationships will be tested by direct measurements of the adhesive dynamics of the T-cell proteins CD2 and CD48. Direct force measurements will quantify the adhesion as a function of the thermodynamic and kinetic properties of the bonds. They will also elucidate how these proteins' structures control both the bond strength and the long-range forces that govern association rates. Lateral interactions between proteins on membrane surfaces may promote the formation of protein assemblies necessary for strengthening adhesive contacts between membranes. The formation of cadherin, CD2 and CD48 domains on isolated membranes and at intermembrane junctions will be investigated by fluorescence dynamics spectroscopy. The resulting impact on adhesion of such protein associations will be quantified by direct force measurements.